ABSTRACT Despite more than three decades of intensive effort, currently no effective anti-RAS therapies have reached clinical application. The history of anti-RAS drug discovery has been marked by missteps and mistakes, due in large part to our incomplete understanding of the full complexities of RAS. There remain many perplexing issues that until recently have been ignored by the field. We argue that if these issues remain unresolved, this will compromise the success of anti-RAS drug discovery. In Project 1, we challenge the perception that ?all RAS mutations are created equal?. Our studies focus on pancreatic ductal adenocarcinoma (PDAC), arguably the human cancer most addicted to mutant KRAS, to pursue three key issues. First, there is the still-prevalent assumption that the highly related RAS proteins (HRAS, KRAS 4A/4B and NRAS) are largely functionally equivalent proteins. Therefore, it is perplexing why there is exclusive mutation of KRAS in PDAC. Does this simply reflect the inability of carcinogenic assault to cause mutational activation of HRAS and NRAS in PDAC? Or alternatively and more provocatively ? despite their significant functional similarities, do mutant HRAS and NRAS not have the capability to drive tumor initiation and progression in the pancreas? Second, a survey of all cancers finds that there are 134 distinct cancer-associated missense mutations found in KRAS, with 99% at one of three mutational hotspots (G12, G13 and Q61) ? do they all cause equivalent perturbations in protein function and have equivalent capabilities to drive cancer development? In PDAC, there is a near-exclusive occurrence of G12 mutations, with G13 and Q61 mutations rare ? does this simply reflect DNA mutation frequencies or do G12 mutations cause distinct perturbations that favor their presence in PDAC? Finally, while there are six possible single base change missense mutations at each hotspot, the frequencies are not uniform, and can exhibit striking cancer-type differences. Do the different amino acid substitutions at each hotspot cause distinct perturbations in protein function that then translate to different capabilities to drive cancer development? We propose three aims that will provide further clarity for these three issues. We will determine if: (1) KRAS G12R is distinct among G12 mutations and exhibits mutation-specific regulation and effector dependencies and driver functions in PDAC; (2) there is a biological basis for why KRAS G13 mutations are rare in PDAC; and (3) KRAS Q61 mutants are biochemically and biologically distinct from KRAS G12 and G13 mutants and have distinct consequences for KRAS protein function in PDAC. We propose that ?all RAS mutations are NOT created equal? and that mutation-specific biochemical and/or signaling properties can be exploited for mutation-selective therapeutic strategies. Project 1 studies are tightly interrelated and highly synergistic with Projects 2-4, in both scientific themes and experimental strategies.